Project 2: Therapeutic Targeting of the Ras Pathway by Nanoparticle Delivery of siRNA Three types of nanoparticles, i.e. LPD, LCP and PRINT, will be used to deliver siRNA to lung cancer cells in a genetically engineered mouse (GEM) model (Kim, co-PI). LPD is a self-assembled, core/membrane nanoparticle, the unique feature of which is the high density ofthe surface grafted PEG chains. Previous work by Huang (co-PI) has shown that LPD accumulates in xenografted human lung tumors at the level of 60-80 % injected dose per g of tissue with minimal uptake by liver and spleen. Three daily i.v. injections of LPD containing siRNA effectively silenced the tumor target gene expression. LCP is similar in structure to LPD, but the core is replaced with calcium phosphate amorphous nanoprecipltate that dissolves in the endosomal acidic pH to release its cargo and swells and bursts the endosomes. The PRINT (DeSimone, co-1) are topdown manufactured nanoparticles In which any carrier and cargo materials can be readily loaded. Dr. DeSimone has shown that siRNA can be successfully loaded in PRINT and delivered to tumor cells for gene silencing. A salient feature of the project is the KRas driven lung cancer GEM. sIRNAs designed against Ras and Ras effector pathways will be tested and delivered as therapeutics. This is particularly significant, because Ras is considered an undruggable target by conventional medicinal chemistry approaches. Furthermore, gemcitabine mono (or di-) phosphate (GMP or GDP), metabolites of gemcitabine will be formulated in LCP nanoparticles and delivered to the tumor cells by itself or together with siRNA. This is a novel approach that takes advantage of the large cargo capacity of these nanoparticles. The project is milestone driven in that by the end of the year 3, at least one nanoparticle formulation will be identified for therapy, pharmacokinetics and toxicity studies In GEM. The formulation should be ready for detailed characterization and toxicity studies in collaboration with Nanotechnology Characterization Lab at NCI for further translational development.